Bcl-2 is a membrane protein that functions as an inhibitor of cell apoptosis. Aberrant levels of bcl-2 are associated with many human diseases including cancer, neurological disorders, and cardiovascular diseases. Effective modulation of bcl-2 expression offers promise for the treatment of these diseases. We have found that the human bcl-2 gene contains a GC-rich proximal promoter region that can form two stable intramolecular G-quadruplex DNA secondary structures using overlapping guanine-rich DNA sequences. This GC-rich region contains a binding site of the WT1 protein which has been shown to be a negative regulator of the bcl-2 gene expression. We have recently developed a screening assay of small molecule compounds that can selectively bind the bcl-2 promoter G-quadruplex structures. Intriguingly, these compounds have been shown to upregulate the bcl-2 gene transcription. The hypothesis to be tested is that stabilization of the bcl-2 promoter G-quadruplex secondary structure(s) with small molecules upregulates bcl-2 gene transcription by inhibiting the binding of the negative regulator WT1 protein. A G-quadruplex DNA secondary structure has been demonstrated to be a transcriptional silencer element in the proximal promoter region of the human c-Myc gene and is amenable to small molecule drug targeting. The G-quadruplexes formed in the promoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-Myc promoter in that their formation could serve to modulate gene transcription. However, the complexity of the G-quadruplex structures in the bcl-2 promoter is higher than is the case for the c-Myc promoter. The presence of two interchangeable G-quadruplexes overlapping in the region of the G-rich strand is likely to be important for the precise regulation of bcl-2 gene transcription, as each G-quadruplex may bind to different proteins leading to different gene modulation, in a manner analogous to the genetic switch in the bacteriophage lambda controlled by the interactive Cro and Repressor proteins, whose operator regions (ORs) overlap with each other's promoter regions and thereby inhibit each other's transcription. In this proposal we aim to determine the biological roles and molecular structures for the bcl-2 promoter G-quadruplex structures. Our primary approach, high-field NMR spectroscopy, represents a major tool for determination of DNA secondary structures under physiological conditions, due to the difficulty of crystallization of such structures. Our long-term goal is to use structure-based rational design to develop small molecule compounds that specifically target the bcl-2 promoter G-quadruplex structures and effectively modulate bcl-2 gene expression. Specifically, we plan to 1) determine the functional significance of the two interchangeable bcl-2 promoter G-quadruplexes in bcl-2 gene regulation and how binding of G-quadruplex-stabilizing compounds affects the regulation of the bcl-2 gene; and 2) to determine the structures of the two interchangeable bcl-2 promoter G-quadruplexes and their complexes with G-quadruplex-stabilizing compounds.